The Gram stain is a well known and widely used microbiological technique used for classifying and identifying bacteria. In hospitals and clinical laboratories, the Gram stain is one of the most frequently performed microbiological diagnostic tests. The Gram stain essentially divides the bacterial world into two distinct classifications: Gram positive bacteria and Gram negative bacteria. Separation into these two classes is based on differences in the structure of the bacterial walls.
The conventional and most commonly used method of the Gram staining technique requires the use of four separate reagents. The reagents consist of a primary staining solution usually containing crystal violet dye, a mordant solution usually containing an iodine-potassium iodide solution, a decolorizing agent usually a solution of acetone and alcohol, and a counter-staining solution usually containing safranin. The conventional method involves the steps of: (1) treating a slide having bacteria affixed upon it with the principal stain, waiting 30-60 seconds; (2) rinsing the slide with water; (3) treating the slide with the mordant solution, waiting 30-60 seconds; (4) rinsing slide with water; (5) decolorizing the slide with acetone and alcohol solution, waiting 30-60 seconds; (6) rinsing the slide with water; (7) counter-staining the slide with the safranin or fuchsin solution for 30-60 seconds and; (8) rinsing the slide with water. Only after these steps are completed can the bacteria affixed to the slide be observed and analyzed to determine Gram status. Typically, Gram positive bacteria will appear purple in color, while Gram negative bacteria will appear reddish, pink in color.
It should be obvious, from the above description of the conventional method of Gram staining, that the conventional method of Gram staining is a very involved, time consuming, and labor intensive process. Each additional step introduces the possibility of error which can lead to an incorrect or inconclusive identification of the bacteria. A Gram stain which results in an inconclusive or incorrect identification of a bacterial specimen must either be repeated or have other diagnostic tests performed to more positively identify the bacterial specimen. In either case, the initiation of effective treatment is delayed when conclusive bacterial identification is not obtained.
For years, technicians using the conventional Gram staining method have been plagued by these inconsistent results. Much of the inconsistency is directly attributable to the conventional method itself. For instance, the separate decolorizing step is problematic in that it is difficult to achieve the proper amount of decolorization of the bacteria. In other words, decolorization, the step of removing the fixed crystal violet stain from the Gram negative organisms, is prone to inconsistency. The inconsistency stems from difficulties associated with the amount of decolorizing solvent applied to bacteria and the amount of time the decolorizing solvent is allowed to contact the bacteria. If the decolorizing solvent is applied in large volumes or is applied too vigorously, the bacteria in the sample may be under-stained, thereby potentially yielding an inconsistent or incorrect result i.e., a truly Gram positive bacterium may appear to be a Gram negative bacterium. If, on the other hand, a bacterial sample is not adequately decolorized, the bacteria in the sample may be over-stained, thereby potentially yielding an inconsistent or incorrect result i.e., truly Gram negative bacterium may appear to be a Gram positive bacterium.
Using the conventional four reagent Gram staining method, certain bacteria may appear as to be both Gram positive and Gram negative. These bacteria are referred to in the art as Gram variable bacteria. Gram variable bacteria present a unique challenge to the technician in that the results of a Gram stain on a pure culture of one of these bacteria could yield both Gram positive and Gram negative bacteria or the bacteria may be stained in such a way that it is impossible to make a determination of the bacteria's Gram status. Instead of Gram positive bacteria appearing purple in color and Gram negative bacteria appearing reddish, pink in color, the bacteria may appear brownish purple or simply just brown. A result like this makes identifying and classifying a bacterial sample very difficult, as is described below.
In addition to inconsistency, the conventional Gram staining method takes more time to perform i.e., an additional reagent adds at least another one minute application period and an additional water wash. Also, the conventional Gram staining method is more costly, since costs typically increases the more reagents that are involved and there is a cost associated with a technician or a machine performing the additional manipulations connected with an additional reagent.
U.S. Pat. No. 4,857,459 to Reuben discloses a method for staining acid-fast bacilli which utilizes a decolorizing counter-stain solution containing methylene blue, ethyl alcohol, potassium hydroxide, glycerol, acetic acid, and polyvinylpyrollidone. However, the decolorizing counter-stain solution and method for its use disclosed in the Reuben '459 patent are for performing an acid-fast stain and not for performing a Gram stain.
There have been other attempts to streamline the conventional Gram staining method and reagents. U.S Pat. No. 4,916,061 to Di Ianni discloses a Gram staining method in which the number of individual reagents was reduced by combining the mordant and the decolorizer into a single reagent comprised of an iodine-iodide salt complex and an alcohol solvent. The Di Ianni '061 patent sought to both streamline the Gram staining process and also to provide a more stable, longer lasting mordant solution. Unfortunately, this method provides inadequate results and, therefore, has no real utility, as described in detail below. It, therefore, follows that simply combining reagents from the conventional Gram staining method does not necessarily yield an improved method or unexpected results. Additionally, merely combining the counter-stain and the decolorizer without adjusting the pH to approximately 4.5 does not provide a pronounced improvement over prior art Gram staining methods.
The present invention not only streamlines the Gram staining method, it yields the added benefits of improved accuracy and performance by eliminating the certain long standing problems such as over/under decolorizing and variability.